CXCR4 is the only known receptor for SDF 1 and has a high affinity

To confirm that these methods are capable of identifying functional hap ploinsufficency in a model of BRCA1 inherited germline mutation, cell lines of known HRR status were assessed. Lastly, primary AML samples were used to demonstrate the ability to quantify activation of these pathways in primary cancer cells. Flow sample and specimen description Cell lines 17-AAG Geldanamycin Individual cell lines harboring mutations in ATM, BRCA2 or BRCA1 were used in the study. Following the suppliers instructions, cell lines obtained from ATCC were maintained in RPMI 1640 supplemented with 10% FCS, and cell lines obtained from Coriell Cell Repositories were maintained in complete RPMI 1640 supplemented with 15% FCS. Cell line panel 1 was used to establish tools for measur ing ATM dependent DNA damage responses.<br><br> ATM wild type cell lines, were compared with an ATM cell line and two ATM cell lines for measurement of etoposide induced DDR readouts. Cell line panel 2 was used to detect differences between BRCA2. BRCA1, and BRCA1samples in response to PARPi treatment. To model BRCA2 status, an Epstein Barr Virus transformed B lymphocblast cell line from a Fanconis Anemia 17-DMAG Alvespimycin patient with homozygous BRCA2FANCD1 mutation was used. To model heterozy gous BRCA1 mutation, 5 EBV transformed B lymphoblast cell lines from pa tients with BRCA1 mutated familial breast cancer were used. Similarly, to model wild type BRCA1 status, 5 EBV transformed B lymphoblast cell lines from breast cancer patients whose tumors are negative for BRCA1 mutations or from healthy donors were evaluated.<br><br> Patient samples AML samples consisted of either peripheral blood mo nonuclear cell or bone marrow mononuclear cell specimens obtained from A66 pediatric or adult patients with AML. Mononuclear cells were purified by ficoll centrifugation then cryopreserved in 90% FBS, 10% DMSO. In accordance with the Declaration of Helsinki, all patients consented to the collection of biospecimens for biology studies. Sample processing and instrument details SCNP assay SCNP assays were performed as described previously. Aliquots of cryopreserved cells were thawed at 37 C, washed, resuspended in RPMI 1640 medium supplemented with 60% fetal bovine serum, and live mononuclear cells isolated via ficoll density gradient. After a second washing step with RPMI 1640 60% FBS, cells were washed in RPMI 1640 10% FBS, counted, filtered, re suspended in RPMI 1640 10% FBS, then aliquoted and rested for 30 minutes at 37 C before addition of therapeutic agents.<br><br> For all condi tions, following incubation with drugs, cells were stained with amine aqua viability dye to distinguish non viable cells, fixed with 1. 6% para formaldehyde for 10 minutes at 37 C, pelleted, perme abilized with 100% ice cold methanol, and stored at 80 C. For antibody staining, cells were washed with FACS buffer, pelleted, and stained with unlabeled antibody cocktails followed by fluorochrome conjugated goat anti mouse or goat anti rabbit secondary antibodies, then blocked with normal rabbit serum and normal mouse serum and stained with cockails of fluorochrome conjugated anti bodies. Cocktails included antibodies against cell surface markers for cell gating of AML cells and up to 3 antibodies against intracellular signaling molecules for 6 8 color flow cytometry assays.